IRBIT activates NBCe1-B by releasing the auto-inhibition module from the transmembrane domain.

The electrogenic Na+ /HCO3 - cotransporter NBCe1-B is widely expressed in many tissues in the body. NBCe1-B exhibits only basal activity due to the action of the auto-inhibition domain (AID) in its unique amino-terminus. However, NBCe1-B can be activated by interaction with the IP3R-binding protein released with inositol 1,4,5-trisphosphate (IRBIT). Here, we investigate the molecular mechanism underlying the auto-inhibition of NBCe1-B and its activation by IRBIT. The IRBIT-binding domain (IBD) of NBCe1-B spans residues 1-52, essentially consisting of two arms, one negatively charged (residues 1-24) and the other positively charged (residues 40-52). The AID mainly spans residues 40-85, overlapping with the IBD in the positively charged arm. The magnitude of auto-inhibition of NBCe1-B is greatly decreased by manipulating the positively charged residues in the AID or by replacing a set of negatively charged residues with neutral ones in the transmembrane domain. The interaction between IRBIT and NBCe1-B is abolished by mutating a set of negatively charged Asp/Glu residues (to Asn/Gln) plus a set of Ser/Thr residues (to Ala) in the PEST domain of IRBIT. However, this interaction is not affected by replacing the same set of Ser/Thr residues in the PEST domain with Asp. We propose that: (1) the AID, acting as a brake, binds to the transmembrane domain via electrostatic interaction to slow down NBCe1-B; (2) IRBIT activates NBCe1-B by releasing the brake from the transmembrane domain.